Irreversible spectrin-haemoglobin crosslinking in vivo: a marker for red cell senescence.
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Centrifugation, Density Gradient↗
Biomedical subjects
L M Snyder
Publications and source records attributed to L M Snyder.
Spectrin-haemoglobin crosslinkages associated with in vitro oxidant hypersensitivity in pathologic and artificially dehydrated red cells.
Protein crosslinkages are apparent at 215 000-250 000 daltons in electrophoregrams of membranes from hydrogen peroxide treated erythrocytes (British Journal of Haematology, 48, 435, 1981). Hydrogen peroxide is also capable of inducing crosslinkages of identical molecular weights in stage I and II (red) ghosts and in a mixture of purified spectrin and haemoglobin, but not in white ghosts or in either spectrin or haemoglobin alone. Autoradiographic studies using 14C-methaemoglobin and 32P-spectrin confirm the involvement of spectrin and haemoglobin in this reaction. The alpha chains of both proteins are more reactive than the corresponding beta chains: 3 times more reactive in the case of spectrin and 10 times more reactive in haemoglobin. The reaction is almost totally inhibited by NaCN and partially inhibited by N-ethylmaleimide. Direct addition of malondialdehyde to a spectrin-haemoglobin mixture does not result in protein crosslinkage. Metabolic depletion (40 h), in vivo ageing and sucrose dehydration of fresh, normal cells enhance the reaction considerably, whereas in vitro rehydration of xerocytes normalizes their H2O2 sensitivity.
Erythrocyte Membrane↗
Increased sensitivity of isolated alpha subunits of normal human hemoglobin to oxidative damage and crosslinkage with spectrin.
After peroxidation, alpha and beta subunits of normal human hemoglobin demonstrated a significant differential reactivity in their ability to form methemoglobin subunits and irreducible crosslinkages with spectrin. The alpha subunits formed crosslinks with spectrin in the absence of exogenous hydrogen peroxide, whereas both the beta subunit and the intact hemoglobin molecule required a minimum of 40 and 4 microM peroxide, respectively, in order to form these crosslinks. Changes in the amount of methemoglobin occurred at much higher concentrations of hydrogen peroxide for the beta hemoglobin subunit (100 microM H2O2) than for the alpha subunit (0.1 microM H2O2). A possible explanation for the existing reactivity between each of the two hemoglobin subunits and spectrin is considered and discussed. In our notation, alpha subunit = alpha SH and the beta subunit = beta SH.
Cross-Linking Reagents↗
A calorimetric comparison of structural transitions of erythrocytic ghosts from normal individuals and from patients with muscular dystrophy.
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Calorimetry, Differential Scanning↗
Characteristics of peripheral blood monocytes in hereditary xerocytosis and spherocytosis.
Peripheral blood monocytes isolated from patients with congenital hemolytic anemia, hereditary xerocytosis and spherocytosis, demonstrated in vivo engulfment of red cell and platelet fragments. In addition, morphometric studies performed on these monocytes showed an increase in cytoplasmic/nuclear ratio as well as lysosome and phagosome volumes. The production of carbon dioxide from glucose-1-14C in abnormal monocytes was increased (15-80%) but the intracellular values of beta-glucuronidase and esterase activity were similar to control monocytes. Monocyte locomotion assessed in the presence of chemotactic stimuli was found significantly increased (73 +/- 12 monocytes/oil immersion fields vs. 46 +/- 5 for control monocytes). We concluded that the monocytes in hemolytic anemias associated with increased in vitro red cell fragmentation have some features resembling the 'stimulated' monocytes and that this alteration may be due to red blood cell fragment ingestion.
Anemia, Hemolytic, Congenital↗
Altered red blood cell surface area in hereditary xerocytosis.
Hereditary xerocytes appear larger than normal red cells in scanning electron micrographs and exhibit a higher ghost packing volume. The major chemical components--protein, phosphorus, cholesterol and sialic acid--are increased uniformly, as are all polypeptides visible on gel electrophoresis patterns of xerocyte membranes. These data are consistent with a xerocyte surface area 15 to 25% above normal. Certain clinical anomalies common to this disorder, including unexpectedly low reticulocyte count and 2,3-diphosphoglycerate level, are discussed in the light of the present findings.
Cholesterol↗
Red cell membrane response to hydrogen peroxide-sensitivity in hereditary xerocytosis and in other abnormal red cells.
Osmotically resistant red cells associated with some haemolytic anaemias, including hereditary xerocytosis, sickle-cell disease and beta thalassaemia minor, are more sensitive than normal red cells to exogenous in vitro hydrogen peroxide (H2O2). This sensitivity is manifested by a rapid loss of intracellular potassium, shape change, protein aggregation, and methaemoglobin formation at lower concentrations of H2O2 (225 microM) than are required to induce similar effects in normal red cells (450 microM). Malonyldialdehyde (MDA) formation occurs later than the other effects and can be inhibited by the antioxidant, butylated hydroxytoluene (BHT), without affecting protein aggregation or potassium leak. Incubation of normal red cells directly with MDA induces protein aggregation, but only after 1 h of incubation. Although nystatin-sucrose treated normal cells which are dehydrated with altered cation content, and therefore osmotically resistant, do not display abnormal H2O2 hypersensitivity as manifested by excessive potassium permeability, they do show an increase in methaemoglobin formation and protein aggregation similar to xerocytes. These data indicate that membrane protein cross-linking occurring immediately following H2O2 exposure seems independent of either the sulfhydryl or MDA mechanisms, and that the membrane permeability of the abnormal red cells predisposes them to oxidative damage.
Anemia, Hemolytic, Congenital↗
Diverse familial malignant tumors and Epstein-Barr virus.
Continued monitoring of a family for new malignant tumors has revealed diverse immunological and neoplastic disorders during a 15-year period. In 1966, the proband developed lymphoma. In 1975, his antibody titers to Epstein-Barr virus (EBV) became elevated, and again, he developed a malignant lymphoma. He also had borderline hypo-immunoglobulin A, died of glioblastoma multiforme in 1977, and at autopsy, had adenomatous colonic polyps. His eldest brother has normal immunoglobulin levels, but developed immune thrombocytopenia in 1973 and had elevated EBV antibody titers in 1980. Another brother had hypo-immunoglobulin A, thymoma in 1965, and adenomas and adenocarcinoma of the colon. Two other brothers succumbed to glioblastoma in 1968 and 1969. The father of the proband had bronchiectasis in 1952, hypo-immunoglobulin M documented in 1972, and elevated EBV antibody titers 5 years preceding development of a malignant lymphoma. The latter contained 10 EBV genome equivalents/cell by EBV viral DNA/DNA reassociation kinetics analysis. The proband's grandmother had died of an immunoglobulin G-secreting myeloma in 1977, and his grandfather had borderline low immunoglobulin M, elevated EBV antibody titers, and hypopharyngeal carcinoma in 1980. Predisposition to oncogenesis in this family was probably inherited.
Adenocarcinoma↗
Contribution of whole cell and cytoplasmic polypeptides to apparent red cell membrane alterations.
We have compared densitometric tracings of whole cell, cytoplasmic and membrane polypeptide electrophoretic patterns in an attempt to distinguish atypical partitioning from intrinsic membrane polypeptide changes occurring as a result of reticulocyte enrichment, metabolic depletion, N-ethylmaleimide treatment and hereditary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in tary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in the partitioning of polypeptides between membranes and supernatant (cytoplasm) at hemolysis. The power of this approach in resolving the sources of apparent red cell membrane protein alterations is demonstrated in studies with hereditary xerocytes. Suggested altered partitioning of these cells described earlier (Sauberman, N., Fortier, N.L., Fairbanks, G., O'Connor, R.J. and Snyder, L.M. (1979) Biochim. Biophys. Acta 556, 292-313) is further documented and found to be unrelated to the younger cell population or slight metabolic depletion that occurs during the washing of xerocytes prior to hemolysis.
Cytoplasm↗
Red blood cell associated IgG in normal and pathologic states.
We studied the anti-IgG-induced agglutination of both normal and abnormal red blood cells (RBC) using a sensitive, automated antiglobulin test. Normal RBC agglutinated strongly with anti-IgG antibody, indicating that IgG was present on the erythrocyte membrane. Young RBC, recovered by centrifugation from a normal RBC population, agglutinated with anti-IgG less than the old cells, suggesting that immunoglobulin G accumulated gradually on the RBC membrane in vivo. The degree of anti-IgG-induced RBC agglutination correlated negatively with the reticulocyte count and positively with the concentration of plasma IgG. RBC from patients with hypogammaglobulinemia appeared to have a low subnormal quantity of membrane-bound IgG, whereas the reverse was the case in hypergammaglobulinemia. During hemolytic episodes, RBC of patients with hereditary spherocytosis agglutinated poorly with anti-IgG, apparently due to predominance of young RBC. RBC of patients with nonspherocytic. Coombs-negative, nonimmune hemolytic anemia usually also agglutinated poorly with anti-IgG. However, in some cases of active hemolytic anemia, decreased agglutination with anti-IgG was not observed, suggesting that these young RBC had increased amounts of membrane-bound IgG.
Agammaglobulinemia↗
Defense of mouse red blood cells against oxidative damage by phenylhydrazine. Glutathione peroxidase and catalase deficiency.
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Acatalasia↗
Increased erythrocyte lipid peroxidation in hereditary xerocytosis.
Xerocytosis is a chronic hemolytic anemia with abnormal membrane function manifested by an increase in passive potassium permeability. Xerocytes demonstrate a greater susceptibility to hydrogen peroxide manifested by the production of malondialdehyde (MDA). Xerocyte membrane phospholipid and fatty acid analysis is normal except for a slight increase in phosphatidyl choline, a commensurate decrease in sphingomyelin, as well as a decrease in linoleic acid. Metabolism and glutathione stability are normal as well as plasma vitamin E levels in patients with xerocytosis. The increased susceptibility to oxidant stress is exaggerated in the "older aged" xerocyte population and correlated well with decreased intracellular potassium concentration.
Anemia, Hemolytic, Congenital↗
Red cell membrane in hemolytic disease. Studies on variables affecting electrophoretic analysis.
Significant alterations in the spectrin: band 3 and band 4.1a: band 4.1b ratios and an occasional decrease in the peak height of band 4.2 with respect to band 4.1 were found in electrophoretic patterns of red cell membranes from patients with hereditary xerocytosis. Electrophoretic comparison of whole cell, cytoplasm and membrane polypeptides implied that atypical partitioning at hemolysis could account for some, but not all, of the alterations seen in membrane patterns of xerocytes. A decrease in band 4.2 peak height as well as a variation in the profile of band 3 were produced in controls by specific manipulations of the electrophoresis protocol. Metabolic depletion of normal cells produced the type of alterations in bands 3 and 4.1 found in xerocyte membranes, whereas Heinz body production, addition of calcium to the hemolysis buffer and incubation of membranes in detergent under conditions designed to promote proteolysis did not. The presence of a higher peak height of band 4.1b with respect to that of band 4.1a in membranes of patients with various other red cell disorders correlated with an increase in the percentage of reticulocytes in peripheral circulation. The appearance of both band 3 and 4.1 abnormalities in the patterns of control cells which had been enriched in young cells by density gradient centrifugation suggested that these alterations in hemolytic disease are related to the predominance of young cells in the population.
Adenosine Triphosphate↗
Catecholamines in adult iron deficiency patients.
Patients with iron deficiency whether uncomplicated or associated with other types of anemias, had plasma catecholamine levels which were significantly increased above normal controls. Patients with a variety of other anemias had no significant increase in catecholamine levels. Plasma catecholamine levels in uncomplicated iron deficient patients approached normal values as early as 3 h following oral FeSO4.
Anemia↗
Metabolic dependence of protein arrangement in human erythrocyte membranes. I. Analysis of spectrin-rich complexes in ATP-depleted red cells.
The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. We investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing two-dimensional SDS-polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited (1) an increase in components 4.5 and 8 and globin subunits, (2) a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9, and (3) a large molecular weight (greater than 10(6) daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH levels with glucose, adenine, and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase-deficient fresh erythrocytes that showed marked GSH depletion but preserved greater than 70% of the original ATP level. However, membranes of these cells did not contain the greater 10(6) dalton aggregate with a high spectrin to band 3 ratio. We concluded that the formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.
Adenosine Triphosphate↗
Fragmentation and myelin formation in hereditary xerocytosis and other hemolytic anemias.
Erythrocytes from a heterogeneous group of hemolytic anemias have been found to release acetylcholinesterase-enriched fragments and show myelin forms during ATP depletion in vitro. The highest amount of fragmentation was found in hereditary spherocytosis and xerocytosis, two inherited membrane defects. Our data suggest ATP depletion plays a role in producing fragmentation or myelin forms. The addition of external CaCl2 1 mM had no effect on the degree of fragmentation. However, propranolol hydrochloride, a cationic anesthetic that does not prevent ATP depletion, inhibited fragmentation and the appearance of myelin forms in both hereditary spherocytes and xerocytes. A more detailed study of the xerocyte fragments showed that they had the same protein composition as those from normal red cells, primarily integral membrane proteins and glycoproteins. The red cells from patients with PNH and G6PD deficiency had the shortest survival in vivo (51Cr) and produced the smallest amount of fragmentation and myelin forms in vitro, whereas xerocytosis with only mild to moderate hemolysis in vivo was associated with the highest amount of myelin forms and membrane fragments in vitro.
Acetylcholinesterase↗
Partition of catalase and its peroxidase activities in human red cell membrane: effect of ATP depletion.
Partititon of catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase EC 1.11.1.6) and peroxidase (donor:hydrogen-peroxide oxidoreductase EC 1.11.1.7) activities between the red cell membrane and the cytosol were studied under various experimental conditions. A small but significant amount of catalase (1.6%) was retained on human red cell membranes prepared by hemolysing washed red cells with 30 volumes of 10 mM Tris buffer, pH 7.4. Membrane -bound catalase had a relatively higher peroxidase activity than the soluble enzyme fraction. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the solubilized membranes demonstrated catalase to be a single band with a molecular weight of 60 000. Membranes prepared from adenosine triphosphate-depleted red cells depicted a two to three-fold increase in catalase activity, as well as an increase in 60 000 molecular weight band on polyacrylamide gel electrophoresis. The extra amount of retained catalase was a less efficient peroxidase than found in fresh membranes. The binding of catalase to ATP-depleted red cell membranes was dependent upon both pH and hemolysing ratio. Red cells incubated at pH 7.1 demonstrated a decrease in bound catalase, as did membranes prepared from red cells hemolysed at 1:100 dilution. beta-Mercaptoethanol decreased the catalase activity in the membranes and increased the odianisidine peroxidase activity without any significant effect on the 60 000-dalton band.
Adenosine Triphosphate↗
Effect of procaine HCLl on ATP: calcium-dependent alterations in red cell shape and deformability.
Procaine hydrochloric acid, a cationic anesthetic, although unable to prevent the effect of calcium ionophore A23187 on erythrocytes, inhibited the discocyte--echinocyte transformation, increased viscosity, and decreased filterability of red cells undergoing ATP depletion. The effects were abolished by washing ATP-depleted, procaine HCl-treated red cells prior to these determinations. Procaine HCl had no effects on volume, incubated osmotic fragility, or monovalent cation composition of ATP-depleted red cells. The drug increased 45Ca uptake by ATP-depleted red cells but did not change the fraction of membrane-bound calcium. Sodium dodecyl sulfate acrylamide gel electrophoresis of membrane proteins from ATP-depleted red cells revealed formation of high molecular weight protein complexes, which were not formed when biconcave shape and ATP content were maintained by incubation with adenine (0.54 mM) and inosine (12.7 mM); Formation of these complexes was not prevented when the biconcave shape was maintained by procaine HCl. It was concluded that the maintenance of the biconcave shape and normal deformability during ATP depletion by procaine HCl was not related to a displacement of membrane-bound calcium and inhibition of ATP-dependent rearrangement of red cell membrane proteins.
Adenosine Triphosphate↗